An ADP-ribosylation factor (ARF), a 20 kDa guanine nucleotide-binding protein that enhances cholera toxin ADP-ribosyltransferase activity, and the a subunit of Go (Go(alpha)), a guanine nucleotide-binding protein that is believed to regulate ion channels, have been expressed in an insect cell line (Spodoptera frugiperda, Sf-9) using modified baculovirus vector pVL941, containing inserts encoding Go(alpha) (pVL941-G09) and ARF2 (pVL941-ARF2). As described previously, lysates of pVL941-Go9-infected cells contained two proteins reactive with an anti-serum specific for Go(alpha). One co-migrated on SDS-PAGE with Go(alpha) purified from bovine brain (about 39 kDa), was present in both soluble and particulate fractions of Sf-9 cells and was ADP-ribosylated with [32P]NAD and pertussis toxin (PT) in the absence of exogenous beta-gamma subunits. As shown in the present report, this protein incorporated [3H]myristic acid. The second protein (about 43 kDa) was present only in the 50,000 x g supernatant; it was not ADP-- ribosylated by pertussis toxin or [3H]myristoylated. Sf-9 cells infected with a modified baculovirus, pVL941-ARF2, contained proteins of about 20 and about 21 kDa which reacted with anti-ARF polyclonal antibodies on an immunoblot. The 20 kDa protein incorporated [3H]myristic but not [3H]palmitic acid. Acid methanolysis and subsequent separation of the products on a reversed-phase HPLC-column confirmed that the expressed 20 kDa ARF was myristoylated. The sequence of the amino-terminal 15 amino acids of the unmyristoylated protein corresponded to the sequence derived from the cDNA used in construction of the pVL941-ARF2 vector. Myristoylated and unmyristoylated ARF were found in the 150,000 x g supernatant and membrane fractions of infected insect cells. Myristoylation of ARF was therefore neither necessary nor sufficient for targeting of this protein to the membrane. Myristoylated and unmyristoylated ARF enhanced cholera toxin-catalyzed ADP-ribosylation.